epsilon-COP is a structural component of coatomer that functions to stabilize alpha-COP.
We isolated a novel yeast alpha-COP mutant, ret1-3, in which alpha-COP is degraded after cells are shifted to a restrictive temperature. ret1-3 cells cease growth at 28 degrees C and accumulate the ER precursor of carboxypeptidase Y (p1 CPY). In a screen for high copy suppressors of these defects, we isolated the previously unidentified yeast epsilon-COP gene. epsilon-COP (Sec28p) overproduction suppresses the defects of ret1-3 cells up to 34 degrees C, through stabilizing levels of alpha-COP. Surprisingly, cells lacking epsilon-COP (sec28 Delta) grow well up to 34 degrees C and display normal trafficking of carboxypeptidase Y and KKXX-tagged proteins at a permissive temperature. epsilon-COP is thus non-essential for yeast cell growth, but sec28 Delta cells are thermosensitive. In sec28 Delta cells shifted to 37 degrees C, wild-type alpha-COP (Ret1p) levels diminish rapidly and cells accumulate p1 CPY; these defects can be suppressed by alpha-COP overproduction. Mutant coatomer from sec28 Delta cells behaves as an unusually large protein complex in gel filtration experiments. The sec28 Delta mutation displays allele-specific synthetic-lethal interactions with alpha-COP mutations: sec28 Delta ret1-3 double mutants are unviable at all temperatures, whereas sec28 Delta ret1-1 double mutants grow well up to 30 degrees C. Our results suggest that a function of epsilon-COP is to stabilize alpha-COP and the coatomer complex.
Duden
R
R
Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley 94720, USA.
Kajikawa
L
L
Wuestehube
L
L
Schekman
R
R
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport / Q000235:genetics; D002268:Carboxypeptidases / Q000235:genetics; D043402:Cathepsin A; D002417:Cattle; D020756:Coatomer Protein; D005800:Genes, Fungal; D005804:Genes, Lethal; D016153:Genes, Suppressor; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology; D008969:Molecular Sequence Data; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D013696:Temperature
D020756:Coatomer Protein; D046911:Macromolecular Substances; D008565:Membrane Proteins; D002268:Carboxypeptidases; D043402:Cathepsin A